The present invention relates to the field of molecular biology and the use of recombinant organisms for the production of 1,3-propanediol. More specifically it describes the expression of cloned genes that affect the intracellular transport of vitamin B12 in conjunction with genes that effectively convert a carbon substrate to 1,3-propanediol.
1,3-Propanediol is a monomer having utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds.
A variety of chemical routes to 1,3-propanediol are known. For example, 1,3-propanediol is prepared 1) from ethylene oxide over a catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid; 2) by the catalytic solution phase hydration of acrolein followed by reduction; or 3) from hydrocarbons such as glycerol, reacted in the presence of carbon monoxide and hydrogen over catalysts having atoms from Group VIII of the periodic table. Although it is possible to generate 1,3-propanediol by these methods, they are expensive and generate waste streams containing environmental pollutants.
It has been known for over a century that 1,3-propanediol can be produced from the fermentation of glycerol. Bacterial strains able to produce 1,3-propanediol have been found, for example, in the groups Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. In each case studied, glycerol is converted to 1,3-propanediol in a two-step, enzyme-catalyzed reaction sequence. In the first step, a dehydratase catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HP) and water (Equation 1). In the second step, 3-HP is reduced to 1,3-propanediol by a NAD+-linked oxidoreductase (Equation 2).
Glycerolxe2x86x923-HP+H2Oxe2x80x83xe2x80x83(Equation 1)
3-HP+NADH+H+xe2x86x921,3-Propanediol+NAD+xe2x80x83xe2x80x83(Equation 2)
The 1,3-propanediol is not metabolized further and, as a result, accumulates in high concentration in the media. The overall reaction consumes a reducing equivalent in the form of a cofactor, reduced xcex2-nicotinamide adenine dinucleotide (NADH), which is oxidized to nicotinamide adenine dinucleotide (NAD+).
The production of 1,3-propanediol from glycerol is generally performed under anaerobic conditions using glycerol as the sole carbon source and in the absence of other exogenous reducing equivalent acceptors. Under these conditions, in strains of Citrobacter, Clostridium, and Klebsiella, for example, a parallel pathway for glycerol operates which first involves oxidation of glycerol to dihydroxyacetone (DHA) by a NAD+- (or NADP+-) linked glycerol dehydrogenase (Equation 3). The DHA, following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA kinase (Equation 4), becomes available for biosynthesis and for supporting ATP generation via, for example, glycolysis.
Glycerol+NAD+xe2x86x92DHA+NADH+H+xe2x80x83xe2x80x83(Equation 3)
DHA+ATPxe2x86x92DHAP+ADPxe2x80x83xe2x80x83(Equation 4)
In contrast to the 1,3-propanediol pathway, this pathway may provide carbon and energy to the cell and produces rather than consumes NADH.
In Klebsiella pneumoniae and Citrobacter freundii, the genes encoding the functionally linked activities of glycerol dehydratase (dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon. The dha regulons from Citrobacter and Klebsiella have been expressed in Escherichia coli and have been shown to convert glycerol to 1,3-propanediol.
The biological production of 1,3-propanediol requires glycerol as a substrate for a two step sequential reaction in which a dehydratase enzyme (typically a coenzyme B12-dependent dehydratase) converts glycerol to an intermediate, 3-hydroxypropionaldehyde, which is then reduced to 1,3-propanediol by a NADH-(or NADPH) dependent oxidoreductase. These cofactor requirements are complex and necessitate the use of a whole cell catalyst for an industrial process incorporating this reaction sequence for the production of 1,3-propanediol. A process for the production of 1,3-propanediol from glycerol using an organism containing a coenzyme B12-dependent diol dehydratase is described in U.S. Pat. No. 5,633,362 (Nagarajan et al.). However, the process is not limited to the use of glycerol as feedstock. Glucose and other carbohydrates are suitable substrates and, recently, these substrates have been shown to be substrates for 1,3-propanediol production. Carbohydrates are converted to 1,3-propanediol using mixed microbial cultures where the carbohydrate is first fermented to glycerol by one microbial species and then converted to 1,3-propanediol by a second microbial species. U.S. Pat. No. 5,599,689 (Haynie et al.). For reasons of simplicity and economy, a single organism able to convert carbohydrates to 1,3-propanediol is preferred. Such an organism is described in U.S. Pat. No. 5,686,279 (Laffend et al.).
Some bacteria, such as Salmonella or Klebsiella, are able to synthesize coenzyme B12 to enable a diol or glycerol dehydratase to operate, but other species must transport B12 from outside of the cell. The term xe2x80x9cB12xe2x80x9d is used to refer collectively to coenzyme B12; derivatives of coenzyme B12 where the upper axial 5xe2x80x2-deoxyadenosyl ligand is replaced with another ligand (for example, an aquo-, cyano- or methyl group); and the radical species, cob(II)alamin.
B12 transport into bacteria presents two major problems. First, the B12 molecule is too large for passage through outer membrane porins, thus requiring a specific outer membrane transport system. Second, owing to the scarcity of B12 in the environment, the outer membrane transport system must have a high affinity for B12 and move it into the periplasm for subsequent transport by another system across the inner membrane. For E. coli, which is unable to synthesize the corrin ring of B12, an external supply of B12 is required for growth under certain conditions. These requirements may be modest; when a functional 5-methyltetrahydrofolate-homocysteine methyltransferase (MetH) is present xcx9c25 B12 molecules (methylcobalamin) are required and xcx9c500 coenzyme B12 molecules are needed for ethanolamine ammonia-lyase dependent growth.
Several proteins are required for the transport process. The 66 kDa outer membrane protein BtuB serves as the high affinity (Kd=0.3 nM) receptor for adenosyl-, aquo-, cyano- and methyl cobalamins and the corresponding cobinamides. When grown in the absence of B12 or at low levels ( less than 1 nM) xcx9c200 copies of BtuB are present per cell. However, the growth of cells in media containing high levels of B12 ( greater than 0.1 uM) represses synthesis of BtuB, and even at levels of 5 nM uptake activities are repressed 80-90%. Unlike Salmonella, the E. coli BtuB is not repressed by aerobiosis. Transport into the periplasm requires the interaction of BtuB with a 26 kDa inner membrane protein TonB in an energy-dependent process that also requires co-transport of calcium. In fact, the high affinity binding of B12 to BtuB is calcium dependent and there is evidence for a reciprocal B12 dependent calcium binding site with a Kd for calcium of xcx9c30 nM at pH 6.6 at saturating levels of B12. This affinity for calcium decreases with decreasing pH. TonB uses proton motive force to drive a structural alteration needed for transport. In the absence of TonB, B12 penetrates die outer membrane with very low efficiency. TonB also energizes outer-membrane transport systems for iron, including the FepA and FhuA systems. Thus BtuB competes with these systems for TonB activity. In the absence of protein synthesis, the rate of B12 transport decreases with a half life of xcx9c20 min and is attributable to a loss of TonB activity. Transfer of B12 from BtuB to the periplasmic binding protein is poorly characterized and may involve a protein encoded by the btuF locus, at least in Salmonella.
Transport across the inner membrane is mediated by the BtuC and BtuD proteins encoded by the btuCED operon. BtuC and BtuD resemble transport proteins requiring a periplasmic binding protein, and BtuD has an ATP binding site. Mutant phenotypes of these two genes arc corrected by a modest increase in external B12, and it is thought that the BtuB/TonB system concentrates B12 in the periplasm and fortuitous transport of B12 is thus facilitated into the cytoplasm. BtuE may not be involved in transport and its function is unknown. The btuCED operon appears to be expressed constitutively and is not regulated by the presence of B12 in the growth medium.
The transport pathway can be summarized as an initial binding of B12 to the outer membrane protein BtuB, followed by interaction with the inner membrane protein TonB and the energy-dependent translocation and binding to periplasmic BtuF, and finally transfer to die inner membrane proteins BtuCD and translocation to the cytoplasm.
An important control mechanism for B12 transport is the influence of coenzyme B12 on the levels of the outer membrane protein BtuB. The formation of cellular coenzyme B12 results from the activity of ATP:corrinoid adenosyltransferase, encoded by the btuR gene. As noted above, the presence of B12 in media results in a reduction in BtuB function, but it is important to emphasize that this direct repression is observed only with coenzyme B12 and not with coenzyme B12 precursors, as seen by the addition of various B12 molecules to a btuR-defective strain. Coenzyme B12 precursors supplied in the media may cause repression resulting from its conversion into coenzyme B12. Control appears to alter continuation of message synthesis rather than initiation, so the use of foreign promoters for btuB expression does not necessarily afford protection from regulation by coenzyme B12. An unusual feature of btuB regulation is that repression seems to be as effective when the btuB gene is carried on a multicopy plasmid as when in a single copy. This apparent lack of titration by excess copies of the target sequences suggests a large excess of the repressor (coenzyme B12) in the cell.
By gene fusion studies it appears that both transcriptional and translational control applies to btuB expression and, considered together, these various features suggest a mechanism in which a direct interaction occurs between coenzyme B12 and the mRNA leader. This interaction may induce mRNA folding to stabilize the hairpin thereby blocking ribosome access to the translational start. The requirement for a substantial portion of the btuB transcript in control of its own expression and regulation suggests that post-transcriptional events involving the leader and btuB coding region influence both transcriptional read through and translation initiation. Involvement of transcribed regions in regulation has been documented for attenuation control in amino acid biosynthetic pathways, but the unusual features of btuB regulation are that important regulatory sites are located within the btuB coding sequence and that this regulation affects both transcription and translation.
The problem to be solved by the present invention is how to biologically produce 1,3-propanediol by means of a single recombinant organism containing a coenzyme B12-dependent dehydratase enzyme enhancing the availability of coenzyme B12 to the enzyme by the presence of foreign genes encoding activities responsible for B12 transport.
Applicants have solved the stated problem by providing a single recombinant organism capable of the dehydratase-mediated bioconversion of a fermentable carbon source directly to 1,3-propanediol, where coenzyme B12 availability to the enzyme is enhanced by the presence of B12 transport genes. Preferred substrates are glucose and glycerol from a larger set of substrates including fermentable carbohydrates, single carbon substrates and mixtures thereof.
The present invention provides a process for the bio-production of 1,3-propanediol comprising: (i) contacting a transformed host cell with at least one fermentable carbon source and an effective amount of vitamin B12 whereby 1,3-propanediol is produced, the transformed host cell comprising: (a) at least one copy of a gene encoding a protein having a dehydratase activity; (b) at least one copy of a gene encoding a protein having an oxidoreductase activity; (c) at least one copy of a gene encoding a vitamin B12 receptor precursor protein; (d) at least one copy of a gene encoding a vitamin B12 transport system permease protein; and (e) at least one copy of a gene encoding vitamin B12 transport ATP- or GTP-binding protein; wherein at least one of the genes of (c), (d) or (e) is introduced into the host cell, and (ii) recovering the 1,3-propanediol produced from step (i). The effective amount of vitamin B12 is at a 0.1 to 10.0 fold molar ratio to the amount of dehydratase present.
The invention further provides a transformed host cell expressing a dehydratase enzyme containing (a) at least one copy of a gene encoding a protein having a dehydratase activity; (b) at least one copy of a gene having an oxidoreductase activity; (c) at least one copy of a gene encoding a vitamin B12 receptor precursor (BtuB); (d) at least one copy of a gene encoding a vitamin B12 transport system permease protein (BtuC); and (e) at least one copy of a gene encoding vitamin B12 transport ATP-binding protein (BtuD), wherein at least one copy of the gene of (c), (d), or (e) is introduced into die host cell.
Applicants have provided 25 sequences in conformity with Rules for the Standard Representation of Nucleotide and Amino Acid Sequences in Patent Applications (Annexes I and II to the Decision of the President of the EPO, published in Supplement No. 2 to OJ EPO, 12/1992), with 37 C.F.R. 1.821-1.825 and Appendices A and B (Requirements for Application Disclosures Containing Nucleotides and/or Amino Acid Sequences) with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IYUB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2):345-373 (1984) which are herein incorporated by reference.
SEQ ID NO:1 is the nucleotide sequence for an E. coli btuB, encoding the vitamin B12 receptor precursor protein.
SEQ ID NO:2 is the nucleotide sequence for a Salmonella btuB, encoding the vitamin B12 receptor precursor protein.
SEQ ID NO:3 is the nucleotide sequence for a E. coli btuC, encoding the vitamin B12 transport system permease protein.
SEQ ID NO:4 is the nucleotide sequence for a E. coli btuD, encoding the vitamin B12 transport ATP-binding protein.
SEQ ID NO:5 is the nucleotide sequence for a E. coli btuE, encoding the vitamin B12 transport periplasmic protein.
SEQ ID NO:6 is the nucleotide sequence for dhaB1, encoding the xcex1 subunit of the glycerol dehydratase enzyme.
SEQ ID NO:7 is the nucleotide sequence for dhaB2, encoding the xcex2 subunit of the glycerol dehydratase enzyme.
SEQ ID NO:8 is the nucleotide sequence for dhaB3, encoding the xcex3 subunit of the glycerol dehydratase enzyme.
SEQ ID NO:9 is the nucleotide sequence for dhaT, encoding Klebsiella oxidoreductase enzyme.
SEQ ID NO:10 is the nucleotide sequence for PHK28-26 a 12.1 kb EcoRI-SalI fragment containing the dha operon.
SEQ ID NO:11 is the nucleotide sequence for a multiple cloning site and terminator sequence used in the construction of the expression vector pTacIQ.
SEQ ID NO:12-23 are primers used in the construction of expression vectors of the present invention.
SEQ ID NO:24 is the nucleotide sequence for an insert in pCL1920, used in the construction of the expression cassette for dhaT and dhaB(1,2,3).
SEQ ID NO:25 is the nucleotide sequence for the glucose isomerase promoter sequence from Streptomyces.
The present invention provides a method for biologically producing 1,3-propanediol from a fermentable carbon source in a single recombinant organism. The method incorporates a microorganism containing genes encoding glycerol dehydratase, 1,3-propanediol oxidoreductase, a gene encoding vitamin B12 receptor precursor(BtuB), a gene encoding vitamin B12 transport system permease protein(BtuC), and a gene encoding vitamin B12 transport ATP-binding protein (BtuD). The recombinant microorganism is contacted with a carbon substrate and 1,3-propanediol is isolated from the growth media.
The present method provides a rapid, inexpensive and environmentally responsible source of 1,3-propanediol monomer useful in the production of polyesters and other polymers.
The following definitions are to be used to interpret the claims and specification.
The terms xe2x80x9cvitamin B12 receptor precursorxe2x80x9d, xe2x80x9cBtuBxe2x80x9d or xe2x80x9couter membrane vitamin B12 receptor proteinxe2x80x9d refer to the polypeptide located on the outer membrane of bacteria responsible for the transport of coenzyme B12, cyanocobalamin, aquacobalamin, methycobalamin, and cobinamide from the culture media to the periplasmic space. For the purposes of the present invention BtuB includes, for example, the proteins encoded by the btuB genes of Escherichia coil (GenBank M10112) (SEQ ID NO:1), and of Salmonella typhimurium (GenBank M89481) (SEQ ID NO:2).
The terms xe2x80x9cBtuCxe2x80x9d or xe2x80x9cvitamin B12 transport system permease proteinxe2x80x9d refer to the polypeptide located on the inner membrane of bacteria, that together with BtuD, transports vitamin B12 and coenzyme B12 from the periplasmic space to the cytoplasm. BtuC includes, for example, the polypeptide encoded by the btuC gene of E. coil (GenBank M14031) (SEQ ID NO:3).
The terms xe2x80x9cBtuDxe2x80x9d or xe2x80x9cvitamin B12 transport ATP-binding proteinxe2x80x9d refer to the polypeptide located on the inner membrane of bacteria, that together with BtuC, transports vitamin B12 or coenzyme B12 from the periplasmic space to the cytoplasm. BtuD includes, for example, the polypeptide encoded by the btuD gene of E. coil (GenBank M 14031) (SEQ ID NO:4).
The term xe2x80x9cBtuExe2x80x9d refers to the polypeptide encoded by the btuE gene of E. coli (GenBank M14031) (SEQ ID NO:5) and is an auxiliary component of the transport system.
The terms xe2x80x9cglycerol dehydratasexe2x80x9d or xe2x80x9cdehydratase enzymexe2x80x9d refer to the polypeptide(s) responsible for a coenzyme B12-dependent enzyme activity that is capable of isomerizing or converting a glycerol molecule to the product 3-hydroxypropionaldehyde. For the purposes of the present invention, the dehydratase enzymes include a glycerol dehydratase (GenBank U09771, U30903) and a diol dehydratase (GenBank D45071) having preferred substrates of glycerol and 1,2-propanediol, respectively. Glycerol dehydratase of K. pneumoniae ATCC 25955 is encoded by the genes dhaB1, dhaB2, and dhaB3 identified as SEQ ID NOS:6, 7, and 8 respectively. The dhaB1, dhaB2 and dhaB3 genes code for the xcex1, xcex2, and xcex3 subunits of the glycerol dehydratase enzyme, respectively. Glycerol dehydratase and diol dehydratase enzymes are complexes (with an xcex12xcex22xcex32 subunit composition) that bind coenzyme B12 with a 1:1 stoichiometry.
An xe2x80x9ceffective amountxe2x80x9d of coenzyme B12 precursor (or vitamin B12) will mean that coenzyme B12 precursor (or vitamin B12) is present in the system at a molar ratio of between 0.1 and 10, relative to the dehydratase enzyme.
The terms xe2x80x9coxidoreductasexe2x80x9d or xe2x80x9c1,3-propanediol oxidoreductasexe2x80x9d refer to the polypeptide(s) responsible for an enzyme activity that is capable of catalyzing the reduction of 3-hydroxypropionaldehyde to 1,3-propanediol. 1,3-Propanediol oxidoreductase includes, for example, the polypeptide encoded by the dhaT gene (GenBank U09771, U30903) and is identified as SEQ ID NO:9.
The terms xe2x80x9ccoenzyme B12xe2x80x9d and xe2x80x9cadenosylcobalaminxe2x80x9d are used interchangeably to mean 5xe2x80x2-deoxyadenosylcobalamin. Hydroxocobalamin is the derivative of coenzyme B12 where the upper axial 5xe2x80x2-deoxyadenosyl ligand is replaced with a hydroxy moiety. Aquacobalamin is the protonated form of hydroxocobalamin. Methylcobalamin is the derivative of coenzyme B12 where the upper axial 5xe2x80x2-deoxyadenosyl ligand is replaced with a methyl moiety. The term xe2x80x9ccyanocobalaminxe2x80x9d is used to refer to the derivative of coenzyme B12 where the upper axial 5xe2x80x2-deoxyxe2x80x25xe2x80x2-adenosyl ligand is replaced with a cyano moiety. The terms xe2x80x9cvitamin B12xe2x80x9d and xe2x80x9cB12xe2x80x9d are used interchangeably to refer collectively to coenzyme B12; derivatives of coenzyme B12 where the upper axial 5xe2x80x2-deoxyadenosyl ligand is replaced with another ligand, for example, an aquo-, cyano- or methyl group; and the radical species, cob(II)alamin. The term xe2x80x9ccoenzyme B12 precursorxe2x80x9d refers to a derivation of coenzyme B12 where the upper axial 5xe2x80x2-deoxyadenosyl ligand is replaced. An xe2x80x9ceffective amountxe2x80x9d of coenzyme B12 precursor will mean that coenzyme B12 precursor is present in the system at approximately a 0.1- to 10.0-fold molar ratio to the amount of dehydratase enzyme present.
The terms xe2x80x9cpolypeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably.
The terms xe2x80x9cfermentable carbon substratexe2x80x9d and xe2x80x9cfermentable carbon sourcexe2x80x9d refer to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, glycerol, dihydroxyacetone and one-carbon substrates or mixtures thereof
The terms xe2x80x9chost cellxe2x80x9d or xe2x80x9chost organismxe2x80x9d refer to a microorganism capable of receiving foreign or heterologous genes or multiple copies of endogenous genes and of expressing those genes to produce an active gene product.
xe2x80x9cGenexe2x80x9d refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5xe2x80x2 non-coding sequences) and following (3xe2x80x2 non-coding sequences) the coding sequence. xe2x80x9cNative genexe2x80x9d refers to a gene as found in nature with its own regulatory sequences. xe2x80x9cChimeric genexe2x80x9d refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that arc derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. xe2x80x9cEndogenous genexe2x80x9d refers to a native gene in its natural location in the genome of an organism. A xe2x80x9cforeignxe2x80x9d or xe2x80x9cheterologousxe2x80x9d gene refers to a gene not normally found in the host organism, but which is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A xe2x80x9ctransgenexe2x80x9d is a gene that has been introduced into the genome by a transformation procedure.
The terms xe2x80x9cencodingxe2x80x9d and xe2x80x9ccodingxe2x80x9d refer to the process by which a gene, through the mechanisms of transcription and translation, produces an amino acid sequence. The process of encoding a specific amino acid sequence includes DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated. For example, alterations in the gene sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue (such as glycine), or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes which result in substitution of one negatively charged residue for another (such as aspartic acid for glutamic acid), or one positively charged residue for another (such as lysine for arginine), can also be expected to produce a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on the biological activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity in the encoded products. Moreover, the skilled artisan recognizes that sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1xc3x97SSC, 0.1% SDS, 65xc2x0 C.), with the sequences exemplified herein.
The term xe2x80x9cexpressionxe2x80x9d refers to the transcription and translation to gene product from a gene coding for the sequence of the gene product.
The terms xe2x80x9cplasmidxe2x80x9d, xe2x80x9cvectorxe2x80x9d, and xe2x80x9ccassettexe2x80x9d refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3xe2x80x2 untranslated sequence into a cell. xe2x80x9cTransformation cassettexe2x80x9d refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. xe2x80x9cExpression cassettexe2x80x9d refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in its host.
The terms xe2x80x9ctransformationxe2x80x9d and xe2x80x9ctransfectionxe2x80x9d refer to the acquisition of new genes in a cell after the incorporation of nucleic acid. The acquired genes may be integrated into chromosomal DNA or introduced as extrachromosomal replicating sequences. The term xe2x80x9ctransformantxe2x80x9d refers to the product of a transformation.
The term xe2x80x9cgenetically alteredxe2x80x9d refers to the process of changing hereditary material by transformation or mutation.
The present invention involves the construction of a production organism that incorporates the genetic machinery necessary to convert a fermentable carbon substrate to 1,3-propanediol, in conjunction with genes encoding enzymes needed for the intracellular transport of vitamin B12. The genes involved in 1,3-propanediol production will include a dehydratase gene (typically a glycerol or diol dehydratase) and an oxidoreductase as well as other proteins expected to aid in the assembly or in maintaining the stability of the dehydratase enzyme. These genes may transgenes and introduced into the host cell, or may be endogenous. Genes responsible for the intracellular transport of vitamin B12 will include at least one gene encoding a vitamin B12 receptor precursor protein(BtuB), at least one gene encoding a gene encoding vitamin B12 transport system permease protein(BtuC) and at least one gene encoding vitamin B12 transport ATP-binding protein (BtuD). At least one of these genes will be a transgene and introduced into the production cell. The transformed production cell is then grown under appropriate conditions for the production of 1,3-propanediol.
Recombinant organisms containing the necessary genes that will encode the enzymatic pathway for the conversion of a carbon substrate to 1,3-propanediol may be constructed using techniques well known in the art. In the present invention genes encoding glycerol dehydratase (dhaB) and 1.3-propanediol oxidoreductase (dhaT) were isolated from a native host such as Klebsiella, and together with genes encoding BtuB (btuB), BtuC (btuC), BtuD (btuD), and BtuE (btuE) isolated from native hosts such as E. coli and S. typhimurium are used to transform host strains such as E. coli strain DH5xcex1 or FM5; K. pneumoniae strain ATCC 25955; K. oxytoca strain ATCC 8724 or M5al, S. cerevisiae strain YPH499, P. pastoris strain GTS115, or A. niger strain FS1.
Rationale for dhaB, dhaT
The production of 1,3-propanediol from glucose can be accomplished by the following series of steps. This series is representative of a number of pathways known to those skilled in the art. Glucose is converted in a series of steps by enzymes of the glycolytic pathway to dihydroxyacetone phosphate (DHAP) and 3-phosphoglyceraldehyde (3-PG). Glycerol is then formed by either hydrolysis of DHAP to dihydroxyacetone (DHA) followed by reduction, or reduction of DHAP to glycerol 3-phosphate (G3P) followed by hydrolysis. The hydrolysis step can be catalyzed by any number of cellular phosphatases which are known to be non-specific with respect to their substrates or the activity can be introduced into the host by recombination. The reduction step can be catalyzed by a NAD+(or NADP+) linked host enzyme or the activity can be introduced into the host by recombination. It is notable that the dha regulon contains a glycerol dehydrogenase (E.C. 1.1.1.6) which catalyzes the reversible reaction of Equation 3.
Glycerolxe2x86x923-HP+H2Oxe2x80x83xe2x80x83(Equation 1)
3-HP+NADH+H+xe2x86x921,3-Propanediol+NAD+xe2x80x83xe2x80x83(Equation 2)
Glycerol+NAD+xe2x86x92DHA+NADH+H+xe2x80x83xe2x80x83(Equation 3)
Glycerol is converted to 1,3-propanediol via the intermediate 3-hydroxy-propionaldehye (3-HP) as has been described in detail above. The intermediate 3-HP is produced from glycerol, Equation 1, by a dehydratase enzyme which can be encoded by the host or can introduced into the host by recombination. This dehydratase can be glycerol dehydratase (E.C. 4.2.1.30), diol dehydratase (E.C. 4.2.1.28) or any other enzyme able to catalyze this transformation. Glycerol dehydratase, but not diol dehydratase, is encoded by the dha regulon. 1,3-Propanediol is produced from 3-HP, Equation 2, by a NAD+- (or NADP+) linked host enzyme or the activity can introduced into tie host by recombination. This final reaction in the production of 1,3-propanediol can be catalyzed by 1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or other alcohol dehydrogenases.
The dha regulon is comprised of several functional elements including dhaK encoding dihydroxyacetone kinase, dhaD encoding glycerol dehydrogenase, dhaR encoding a regulatory protein, dhaT encoding 1,3-propanediol oxidoreductase as well as dhaB1, dhaB2, and dhaB3 encoding the xcex1, xcex2 and xcex3 subunits of the enzyme, respectively. Additionally, gene products designated as protein X, protein 1, protein 2, and protein 3 (corresponding to dhaBX, orfY, orfX, and orfW, respectively) are encoded within the dha regulon. While the precise functions of these gene products are not well characterized, the genes are linked to glycerol dehydratase (dhaB) or 1,3-propanediol oxidoreductase (dhaT) and are known to be useful for the production of 1,3-propanediol. Coenzyme B12 that is bound to glycerol dehydratase occasionally undergoes irreversible cleavage to form an inactive modified coenzyme which is tightly bound to the dehydratase. Reactivation of the enzyme occurs by exchange of the bound, modified coenzyme with free, intact coenzyme B12. Protein X and at least one other of protein 1, protein 2, and protein 3 are involved in the exchange process. (See U.S. Ser. No. 08/969,683). In the separate diol dehydratase system, genes designated as ddrA and ddrB, corresponding to the genes encoding protein X and protein 2, respectively, are described to be involved in the exchange process. Mori et al., J. Biol Chem. 272, 32034-32041 (1997).
It is contemplated that glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase may be particularly effective in the conversion of glucose to glycerol, required for the production of 1,3-propanediol. The term xe2x80x9cglycerol-3-phosphate dehydrogenasexe2x80x9d refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P). In vivo G3PDH may be NADH, NADPH, or FAD-dependent. The NADH-dependent enzyme (EC 1.1.1.8) is encoded, for example, by several genes including GPD1 (GenBank Z74071xc3x972), or GPD2 (GenBank Z35169xc3x971), or GPD3 (GenBank G984182), or DAR1 (GenBank Z74071xc3x972). The NADPH-dependent enzyme (EC 1.1.1.94) is encoded by gpsA (GenBank U321643, (cds 197911-196892) G466746 and L45246). The FAD-dependent enzyme (EC 1.1.99.5) is encoded by GUT2 (GenBank Z47047xc3x9723), or glpD (GenBank G147838), or glpABC (GenBank M20938). The term xe2x80x9cglycerol-3-phosphatasexe2x80x9d refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol-3-phosphate and water to glycerol and inorganic phosphate. Glycerol-3-phosphatase is encoded, for example, by GPP1 (GenBank Z47047xc3x97125), or GPP2 (GenBank U18813xc3x9711).
Gene Isolation
Methods of obtaining desired genes from a bacterial genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA may be amplified using standard primer directed amplification methods such as polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for transformation using appropriate vectors.
Alternatively, cosmid libraries may be created where large segments of genomic DNA (35-45 kb) may be packaged into vectors and used to transform appropriate hosts. Cosmid vectors are unique in being able to accommodate large quantities of DNA. Generally, cosmid vectors have at least one copy of the cos DNA sequence which is needed for packaging and subsequent circularization of the foreign DNA. In addition to the cos sequence these vectors will also contain an origin of replication such as ColE1 and drug resistance markers such as a gene resistant to ampicillin or neomycin. Methods of using cosmid vectors for the transformation of suitable bacterial hosts are well described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Typically to clone cosmids, foreign DNA is isolated and ligated, using the appropriate restriction endonucleases, adjacent to the cos region of the cosmid vector. Cosmid vectors containing the linearized foreign DNA are then reacted with a DNA packaging vehicle such as bacteriophage xcex. During the packaging process the cos sites are cleaved and the foreign DNA is packaged into the head portion of the bacterial viral particle. These particles are then used to transfect suitable host cells such as E. coli. Once injected into die cell, the foreign DNA circularizes under the influence of the cos sticky ends. In this manner large segments of foreign DNA can be introduced and expressed in recombinant host cells.
Isolation and Cloning of Genes Encoding Glycerol Dehydratase (dhaB) and 1.3-propanediol Oxidoreductase (dhaT)
Methods for the identification and isolation of dhaB and dhaT were done essentially as described in U.S. Pat. No. 5,686,276 and hereby incorporated by reference. Cosmid vectors and cosmid transformation methods were used within tie context of the present invention to clone large segments of genomic DNA from bacterial genera known to possess genes capable of processing glycerol to 1,3-propanediol. Two 1,3-propanediol positive transformants were analyzed and DNA sequencing revealed extensive homology to the glycerol dehydratase gene (dhaB) from C. freundii, demonstrating that these transformants contained DNA encoding the glycerol dehydratase gene. dhaB and dhaT were isolated and cloned into appropriate expression cassettes for co-expression in recombinant hosts with genes encoding B12 transport functions.
Although the instant invention utilizes the isolated genes from within a Klebsiella cosmid, alternate sources of dehydratase genes include, but are not limited to, Citrobacter, Clostridia, and Salmonella.
B12 Transport Genes
D Rationale for B12 Transport Genes
Adenosyl-cobalamin (coenzyme B12) is an essential cofactor for glycerol dehydratase activity. The coenzyme is the most complex non-polymeric natural product known, and its synthesis in vivo is directed using the products of about 30 genes. Synthesis of coenzyme B12 is found in prokaryotes, some of which are able to synthesize the compound de novo, while others can perform partial reactions. E. coli, for example, cannot fabricate the corrin ring structure, but is able to catalyze the conversion of cobinamide to corrinoid and can introduce the 5xe2x80x2-deoxyadenosyl group.
B12 transport into E. coli may be a limiting factor for the production of a functional DhaB enzyme, in which case increased intracellular availability of coenzyme B12 would be required to optimize glycerol dehydratase activity (and, ultimately, 1,3-propanediol production). This may be achieved by increasing the rate of transport of B12 into the cell. Given the role of coenzyme B12 as a repressor of btuB expression, and the levels of coenzyme B12 required in fermentations, it is likely that B12 transport declines over time due to turnover or dilution of BtuB from cell division. The available pool of free coenzyme B12 in the cell will be influenced by the rate of uptake, the relative affinities of BtuB mRNA and DhaB for coenzyme B12, and the concentrations of the mRNA and DhaB. Since uptake is reduced when using B12 enriched media, an important factor determining whether the uptake mechanism is restored will be partitioning of coenzyme B12 between its regulatory role on btuB expression and DhaB enzyme. This presents an unusual problem of a desired cofactor (coenzyme B12) being responsible for its own limitation. The use of media containing coenzyme B12 precursors in place of coenzyme B12 may alleviate the problem, but this will only be a temporary gain since the transported precursors will be converted to coenzyme B12 by the btuR-encoded adenosyltransferase. One way to circumvent this gene regulation problem is to uncouple BtuB synthesis from coenzyme B12 regulation. Amplification of btuB expression by cloning on multicopy plasmids leads to increased binding of B12 to membranes and increased rates of uptake, and if the btuB native promoter is replaced, will also uncouple synthesis of BtuB from coenzyme B12 regulation.
B12 transport into bacteria requires a specific transport system. Several proteins are required for this transport process. The 66 kDa outer membrane protein BtuB serves as a receptor for adenosyl-, aquo-, cyano- and methyl cobalamins and the corresponding cobinamides. Transport into the periplasm requires the interaction of BtuB with a 26 kDa inner membrane protein TonB in an energy-dependent process. Transport across the inner membrane is mediated by the BtuC and BtuD proteins encoded by the btuCED operon. BtuC and BtuD resemble transport proteins requiring a periplasmic binding protein, and BtuD has an ATP binding site. The transport pathway can be summarized as an initial binding of B12 to the outer membrane protein BtuB, followed by interaction with the inner membrane protein TonB and the energy-dependent translocation and binding to periplasmic BtuF (in S. typhimurium), and finally transfer to the inner membrane proteins BtuCD and translocation to the cytoplasm. Amplification of btuBCED expression by cloning on multicopy plasmids leads to increased binding of B12 to membranes and increased rates of uptake into cells.
Isolation and Expression of the B12 Transport Genes
Expression plasmids that could exist as replicating elements in E. coli were constructed for the four B12 transport genes, btuB, btuC, btuD and btuE. The four genes were isolated by PCR using gene-specific primers and E. coli chromosomal DNA. The four genes were assembled together on expression plasmids. All expression plasmids use a trc promoter for transcription and the native btu ribosome binding sites for translation. Each plasmid also contained either 1) a gene for xcex2-lactamase for selection in E. coli on media containing ampicillin or 2) a gene encoding chloramphenicol acetytransferase for selection on media containing chloramphenicol. Plasmid origins of replication are either ColE1 or p15A.
Host Cells
Suitable host cells for the recombinant production 1,3-propanediol by the coexpression of a gene encoding a dehydratase enzyme and the genes responsible for intracellular B12 transport may be either prokaryotic or eukaryotic and will be limited only by their ability to express active enzymes. Preferred hosts will be those typically useful for production of 1,3-propanediol or glycerol such as Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces and Pseudomonas. Most preferred in the present invention are E. coli, Klebsiella species, and Saccharomyces species.
E. coli, Saccharomyces species, and Klebsiella species are particularly preferred hosts. Strains of Klebsiella pneumoniae are known to produce 1,3-propanediol when grown on glycerol as the sole carbon. It is contemplated that Klebsiella can be genetically altered to produce 1,3-propanediol from monosaccharides, oligosaccharides, polysaccharides, or one-carbon substrates.
Vectors and Expression Cassettes
The present invention provides a variety of vectors and transformation and expression cassettes suitable for the cloning, transformation and expression of genes encoding a suitable dehydratase and genes effecting the intracellular transport of B12 to into a suitable host cell. Suitable vectors will be those which are compatible with the bacterium employed. Suitable vectors can be derived, for example, from a bacteria, a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, a yeast, or a plant. Protocols for obtaining and using such vectors are known to those in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manualxe2x80x94volumes 1,2,3 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)).
Typically, the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5xe2x80x2 of the gene which harbors transcriptional initiation controls and a region 3xe2x80x2 of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.
Initiation control regions or promoters, which are useful to drive expression of the relevant genes of the present invention in the desired host cell, are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, xcexPL, xcexPR, T7, tac, and trc (useful for expression in E. coli).
Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included.
For effective expression of the instant enzymes, DNA encoding the enzymes are linked operably through initiation codons to selected expression control regions such that expression results in the formation of the appropriate messenger RNA.
Transformation of Suitable Hosts and Expression of Genes for the Production of 1.3-propanediol
Once suitable cassettes are constructed they are used to transform appropriate host cells. Introduction into the host cell of the cassette containing the genes responsible for intracellular B12 transport as well as glycerol dehydratase (dhaB), and 1,3-propanediol oxidoreductase (dhaT), either separately or together, may be accomplished by known procedures such as by transformation (e.g., using calcium-permeabilized cells, electroporation) or by transfection using a recombinant phage virus. (Sambrook et al., supra.)
In the present invention, E. coli FM5 containing the genes encoding glycerol dehydratase (dhaB), 1,3-propanediol oxidoreductase (dhaT), BtuB (btuB), BtuC (btuC), BtuD (btuD), and BtuE (btuE) is used to transport vitamin B12 or coenzyme B12 from the media into the cytoplasm to enable glycerol dehydratase to function.
Media and Carbon Substrates
Fermentation media in the present invention must contain suitable carbon substrates. Suitable substrates may include but are not limited to glycerol, dihydroxyacetone, monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose, or mixtures thereof, and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Additionally, the carbon substrate may also be one-carbon substrates (such as carbon dioxide or methanol) for which metabolic conversion into key biochemical intermediates has been demonstrated.
Glycerol production from single carbon sources (e.g., methanol, formaldehyde, or formate) has been reported in methylotrophic yeasts (Yamada et al., Agric. Biol. Chem., 53(2) 541-543, (1989)) and in bacteria (Hunter et al., Biochemistry, 24, 4148-4155, (1985)). These organisms can assimilate single carbon compounds, ranging in oxidation state from methane to formate, and produce glycerol. The pathway of carbon assimilation can be through ribulose monophosphate, through serine, or through xylulose-monophosphate (Gottschalk, Bacterial Metabolism, Second Edition, Springer-Verlag: New York (1986)). The ribulose monophosphate pathway involves the condensation of formate with ribulose-5-phosphate to form a 6 carbon sugar that becomes fructose and eventually the three carbon product glyceraldehyde-3-phosphate. Likewise, the serine pathway assimilates the one-carbon compound into the glycolytic pathway via methylenetetrahydrofolate.
In addition to utilization of one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon-containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth Cl Compd, [Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol., 153(5), 485-9 (1990)). Accordingly, the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing substrates and will only be limited by the requirements of the host organism.
Although it is contemplated that all of the above-mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glycerol, dihydroxyacetone, monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates. More preferred are sugars such as glucose, fructose, sucrose and single carbon substrates such as methanol and carbon dioxide. Most preferred is glucose.
In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for glycerol production. Particular attention is given to Co(II) salts and coenzyme B12 precursors. For example, E. coli and eukaryotes are unable to synthesize coenzyme B12 de novo but are able to utilize coenzyme B12 precursors. Preferred coenzyme B12 precursors are cyanocobalamin and hydroxocobalamin. It is desirable that the amount of coenzyme B12 inside the host cell be approximately equal in molar concentration to the amount of dehydratase enzyme.
Culture Conditions
Typically, cells are grown at 30xc2x0 C. in appropriate media. Preferred growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Malt Extract (YM) broth. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by someone skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 3xe2x80x2:5xe2x80x2-monophosphate, may also be incorporated into the reaction media. Similarly, the use of agents known to modulate enzymatic activities (e.g., sulphites, bisulphites and alkalis) that lead to enhancement of glycerol production may be used in conjunction with or as an alternative to genetic manipulations.
Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the range for the initial condition.
Reactions may be performed under aerobic or anaerobic conditions where anaerobic or microaerobic conditions are preferred.
Fermentations
The present invention may be practiced using either batch, Fed-Batch, or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for 1,3-propanediol production.
The present process is exemplified herein as a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the media is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the media is inoculated with the desired organism or organisms and fermentation is permitted to occur adding nothing to the system. Typically, however, a batch fermentation is xe2x80x9cbatchxe2x80x9d with respect to the addition of the carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. The metabolite and biomass compositions of the batch system change constantly up to the time the fermentation is stopped. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.
A variation on the standard batch system is the Fed-Batch fermentation system which is also suitable in the present invention. In this variation of a typical batch system, the substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen, and die partial pressure of waste gases such as CO2. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Brock, infra.
The method would also be adaptable to continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to media being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology. A variety of methods are detailed by Brock, infra.
Identification and Purification of 1,3-propanediol
Methods for the purification of 1,3-propanediol from fermentation media are known in the art. For example, propanediols can be obtained from cell media by subjecting the reaction mixture to extraction with an organic solvent, distillation, and column chromatography (U.S. Pat. No. 5,356,812). A particularly good organic solvent for this process is cyclohexane (U.S. Pat. No. 5,008,473).
1,3-Propanediol may be identified directly by submitting the media to high pressure liquid chromatography (HPLC) analysis. Preferred in the present invention is a method where fermentation media are analyzed on an analytical ion exchange column using a mobile phase of 0.01 N sulfuric acid in an isocratic fashion.
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.